Targeting of glycoprotein therapeutics

ABSTRACT

Methods of making ligand-decorated polymer conjugates of therapeutic glycoproteins are described. Improved targeting of glycoproteins to specific tissues is achieved by masking the natural carbohydrate and other surface determinants with high molecular weight polymers, such as, e.g., PEG, polysialic acid, etc., which in turn are decorated with target-specific ligands. In some embodiments, acid-labile linkages in such conjugates or rapidly degradable masking groups allow for the intracellular release of the polymer from the glycoprotein, for example, under conditions found in lysosomes.

This application is a continuation of U.S. pat. application No.11/398,949, filed on Apr. 5, 2006 (now U.S. Pat. No. 7,341,720), whichclaims priority to U.S. provisional application No. 60/668,920, filed onApr. 6, 2005, both of which are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

The invention relates to protein therapeutics and, more specifically, toconjugation of such therapeutics with other molecular moieties toachieve tissue-specific targeting in the body, followed by theintracellular release of the biologically active therapeutic at the siteof action, as exemplified by replacement lysosomal enzymes conjugatedwith ligand-decorated polymers and the use of such conjugates fortreatment of lysosomal storage disorders.

BACKGROUND OF THE INVENTION

Tissue-specific targeting of therapeutic proteins to tissues of choicein the body finds application in many medical conditions includingcancer and a number of acquired and inherited disorders. For example, inthe class of diseases called lysosomal storage disorders, an inheriteddeficiency in one or more enzymes which reside in the lysosomes leads tothe accumulation of substrates for those enzymes in the cells. Becauseof tissue-specific patterns of expression and accumulation of thesubstrates within different cells in the body, these disorders result intissue/organ-specific manifestations which vary depending upon thedisorder. These disorders have been found to be treatable by intravenousadministration of the active version of the enzyme deficient in thepatient, a process termed enzyme replacement therapy (ERT). However, theefficacy of ERT varies widely among the different disorders. Althoughthe reasons for this variability are not fully understood, it iscommonly believed to be due to the lack of specific targeting to themost seriously affected tissues.

Most lysosomal proteins are glycoproteins containing one or more N- orO-linked oligosaccharide side chains of high mannose, complex or hybridtype. A number of receptors specific for these sugar residues exist,including among others, those for mannose, galactose (asialoglycoproteinreceptor, ASGPR) and mannose-6-phosphate (cation-independentmannose-6-phosphate receptor, CIMPR). These receptors at least in partmediate the uptake of administered protein into cells. However, thedistribution of these receptors within tissues in the body (e.g., ASGPRexpressed on liver hepatocytes, mannose receptor on cells of thereticulo-endothelial system such as macrophages and Kupffer cells of theliver and CIMPR expressed widely on endothelial cells as well as othercell types) is not optimal for targeting proteins to the tissues whichare most strongly affected. In some cases, modification and/or removalof a portion or all of the oligosaccharide chains through a processtermed remodeling can advantageously improve the ultimatebiodistribution of the proteins to more specifically target the proteinto desired cell types (see, e.g., Furbush et al. Biochimica etBiophysica Acta 673:425-434 (1981), which describes sugar remodeling fora recombinant glucocerebrosidase, imiglucerase (Cerezyme®, GenzymeCorporation, Cambridge, Mass.)). However, complete removal of thecarbohydrate side chains is often counterproductive, since they are alsooften necessary for the solubility and/or intracellular stability of theprotein.

Another difficulty encountered with ERT is the strong immunogenicity ofsome therapeutic proteins as the patient's immune system oftenrecognizes such proteins as foreign and mounts a neutralizing immuneresponse. Thus, a means to reduce the exposure of the therapeuticproteins to the immune system would also be desirable.

Covalent conjugation with polymers such as polyethylene glycol (PEG)generally increases the serum half-life of a number of therapeutics suchas antibodies, interferon, and effector molecules, while also reducingtheir immunogenicity. Although maintaining elevated concentrations ofadministered lysosomal proteins in circulation would similarly beexpected to increase their bioavailability, in the case of lysosomalproteins, conjugation of these proteins with PEG (“PEGylation”) alonedoes not appear to be effective. This may partly be due to the adverseeffect of the conditions in plasma, particularly elevated pH, on enzymestability, and also on the inability of PEG, a neutral hydrophilicpolymer, to influence the relative affinity of the glycoproteins forvarious receptor systems and to introduce any new tissue tropism to theprotein. Thus, an additional means to promote uptake into the lysosomesof cells, and specifically the cells in those tissues in which substratehas accumulated in the body, would be highly desirable. In some cases,this can be achieved by the affinity of the polymer itself for specifictissue types (e.g., PVP-DMMan polymer conjugates for targeting atherapeutic to the kidneys are described in Kamada et al., Nat. Biotech.(2003) 21:399-404). Alternatively, it may be achieved by theintroduction of ligands into the conjugate to promote interaction withtissue-specific receptors to mediate uptake. In the simplest case, suchligands are represented by antibodies against the receptor of choice.However, the larger proteinaceous ligands, such as antibodies, canthemselves be immunogenic, thus posing significant challenges in theclinic.

Additionally, conjugation of a therapeutic protein with high molecularweight polymers may interfere with the activity of the protein at thesite of action in the cell. For example, it has been found that many ofthe lysosomal enzymes, particularly those that act on glycolipidsubstrates, require a cofactor from the class termed saposins for theirenzymatic activity. Saposins are believed to assist in presentation ofthe carbohydrate head group of the substrate to the catalytic site.Thus, conjugation of a high molecular weight polymer to the enzyme mightaffect the enzyme's activity by interfering with interactions withsaposins, thereby lowering the efficacy of the therapeutic. Accordingly,a means to provide for elimination of the polymer from the enzyme in thesite of action would be desirable.

On the other hand, another factor contributing to lowered efficacy ofenzyme replacement therapies is the instability of lysosomal proteinswithin the lysosome, leading to a need for repeated administration. Forexample, Cerezyme® (glucocerebrosidase) is generally administered to apatient having Gaucher's disease on a biweekly basis due to loss of itsactivity after being taken up by target cells. The loss of activity isat least in part due to the action of lysosomal proteases on theprotein, and appending polymers such as PEG can increase the resistanceof proteins to proteolysis. Thus, under certain circumstances, a polymermay serve the additional function of protecting the protein in thelysosomal environment, thereby providing better intralysosomal stabilityof the active protein. Such a strategy may be effective in reducing thefrequency of administration.

Low molecular weight ligands, such as peptides or mono- oroligosaccharides, may be used for targeting a therapeutic protein.However, such ligands often must be present in multiple copies on amacromolecule in order to mediate effective uptake by the cognatereceptor, a condition termed “multivalent display.” Although currentcommercially available heterobifunctional PEGs (e.g., linear moleculescontaining different chemical entities on each terminus) may be used togenerate ternary conjugates, they do not provide for multivalent displayexcept by the attachment of multiple PEG molecules. But such heavymodification often has an adverse effect on enzyme activity.

Therefore, there exists a continuing need to provide proteintherapeutics that allow for target-specific delivery within the body andare sufficiently biologically active upon intracellular uptake.

SUMMARY OF THE INVENTION

The present invention provides ternary conjugates of a therapeuticglycoprotein, a masking moiety, and a targeting moiety. A conjugate ofthe invention includes:

-   -   (1) a therapeutic glycoprotein (G),    -   (2) a masking moiety (M) covalently linked to an oligosaccharide        side chain of the glycoprotein through a first linker (L¹), and    -   (3) a targeting moiety (T) covalently linked to the masking        moiety through a second linker (L²),

wherein the glycoprotein is released from the conjugate under lysosomalconditions.

-   -   In other embodiments, a conjugate includes:    -   (1) a therapeutic glycoprotein (G),    -   (2) a masking moiety (M) covalently linked to an amino acid        residue of the glycoprotein through a first linker (L¹), and    -   (3) a targeting moiety (T) covalently linked to the masking        moiety through a second linker (L²),    -   wherein the glycoprotein is released from the conjugate under        lysosomal conditions.

In some embodiments, the therapeutic glycoprotein is a lysosomal enzyme,such as, e.g., lysosomal enzymes listed in Table 2, including inparticular, glucocerebrosidase, α-galactosidase A, acid α-glucosidase,or acid sphingomyelinase. In some embodiments, the therapeuticglycoprotein is glucocerebrosidase or α-galactosidase A.

In some embodiments, L¹ and/or L² comprise(s) one or more labile groupssuch as, e.g., a hydrazone and/or a disulfide group, that allow for abiologically active glycoprotein to be released at the site of action inthe cells, e.g., in the lysosome.

In some embodiments, the masking moiety is a polymer selected the groupconsisting of polyethylene glycol (PEG), polyvinylpyrrolidone (PVP),polymethacrylate (PMA), polysialic acid (PSA), hyaluronic acid (HA),hydroxy alkyl starches (HAS), albumin, and dextran.

The invention further encompasses methods of making and using theconjugates of the inventions. The conjugates can be used, for example,as pharmaceutical compositions, e.g., for treatment of lysosomal storagedisorders listed in Table 2. In some embodiments, the lysosomal storagedisorder is Fabry, Gaucher, Pompe or Niemann Pick B disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a structural representation of a nonlimiting embodimentof the invention. G, M, and T denote a glycoprotein, a masking moiety,and a targeting moiety, respectively. Linkers L¹ and L² are not shown.

FIG. 2 depicts a scheme for conjugating a therapeutic glycoprotein to ahydrazide-PEG. Terminal sialic acids on the glycoprotein are removed bytreatment with neuraminidase. The exposed terminal galactose residuesare then oxidized to aldehydes by treatment with Dactylium dendroidesgalactose oxidase. Alternatively, aldehydes may be introduced throughoxidation with sodium periodate. The product is then exchanged intobuffer around pH 5.5 and reacted with a hydrazide PEG to form ahydrazone conjugate. The products are purified away from unreacted PEG(e.g., by anion exchange or size-exclusion chromatography).

FIG. 3 shows a scheme for generating a peptide-PEG-glycoproteinconjugate using a heterobifunctional PEG. A heterobifunctional PEG isgenerated by reacting a hydrazide-functionalized PEG with an adaptormolecule (glyoxyl-nipsylethylamide, “GNEA”) containing ahydrazide-reactive glyoxyl aldehyde linked to a thiol reactivefunctional group (nipsylethylamine, “NEA”). This PEG is reacted in thepresence of galactose oxidase with neuraminidase-treated protein toproduce a conjugate in which the PEG is coupled through a hydrazonelinkage to an exposed protein oligosaccharide. This product is purified,and then coupled to a peptide containing a free thiol.

FIG. 4A illustrates the generation of a peptide-PEG-glycoproteinconjugate using thiol/hydrazide chemistry. Aldehyde groups are generatedon the glycoprotein by treatment with periodate or galactose oxidase(GAO) as in FIG. 1. The GAO-treated protein is reacted with an adaptorcontaining a hydrazide and a protected thiol, such as3-(2-pyridyldithio)propionyl hydrazide (PDPH). The adaptor's disulfideis then reduced (e.g., with tris-carboxyethylphosphine, TCEP) to exposethe thiol, which is then reacted with a PEG molecule bearingthiol-reactive moieties.

The resulting conjugate is purified (e.g., by ion-exchangechromatography) and reacted with peptides containing a cysteine moietyto yield a final ternary peptide/PEG/glycoprotein conjugate.

FIG. 4B illustrates conjugation of the glycoprotein through an aminoacid residue. An amino acid residue, in this illustration a lysine, isreacted with S-acetyl-dPEG™_(p) NHS ester to introduce a thiol group onthe glycoprotein. The acetyl-protected thiol is deprotected withhydroxylamine and reacted with an NEA-PEG to produce a disulfide linkedNEA-PEG/glycoprotein conjugate. A targeting moiety bearing a protectedthiol, such as a protein modified with S-acetyl-dPEG™_(q) NHS ester, isdeprotected and reacted with the NEA-PEG/glycoprotein conjugate toproduce the final ternary protein/PEG/glycoprotein conjugate.

FIG. 5 shows the pH-dependent dissociation of a pendant hydrazide PEGconjugate with α-galactosidase A. A pendant hydrazide PEG bearing onaverage eight propionic acid hydrazide groups was conjugated withgalactose-oxidized α-galactosidase as described for FIG. 1. Theconjugate was purified by anion exchange chromatography and exchangedinto buffers of varying pH and incubated overnight at 37° C. asdescribed in Example 2. The amount of protein in PEGylated form relativeto an unincubated control was determined by densitometry of theCoomassie-stained gel following SDS-PAGE.

FIG. 6 shows results of a BIAcore™ analysis for the interaction ofPEG-glycoprotein conjugates with the cation independentmannose-6-phosphate receptor (CIMPR). The extent of unmodifiedα-galactosidase or galactosidase conjugate binding to CIMPR (in RU) wasdetermined using immobilized soluble CIMPR on a dextran-coated chip. Twohydrazide PEGs (10 kDa MW, SunBio) were used for preparing theconjugates by the scheme shown in FIG. 1: (1) a 4-arm star-type PEG(dendrimer; Hz4PEG) and (2) an 8-arm pendant PEG (Hz8PEG). Both werefunctionalized with hydrazide groups either at the PEG termini (Hz4PEG)or internally by random co-polymerization (Hz8PEG). This shows 10-foldhigher concentrations of the Hz4-PEG conjugate were required to obtainthe same degree of receptor binding as obtained with unmodifiedα-galactosidase.

FIG. 7 shows results of a pharmacokinetics study with intravenouslyadministered α-galactosidase and a PEG-α-galactosidase conjugate inFabry mice. An initial blood sample was drawn prior to protein injection(plotted as zero time). Proteins were injected at 1 mg/kg body weight bytail vein and blood withdrawn at 0.5, 1, 2, 4, and 8 hours. Serum wasprepared and assayed for galactosidase activity using a 4MU substrate asdescribed in Example 4.

FIG. 8 shows biodistribution of α-galactosidase (Fabrazyme®) orPEG-Fabrazyme® conjugate in Fabry mice. Proteins were injected at 1mg/kg body weight and the organs harvested 8 hours after injection.Galactosidase activity was determined using a 4MU substrate as describedin Example 4.

FIG. 9 is intracellular uptake levels of variouspeptide-PEG-α-galactosidase conjugates. NEA-PEG conjugates prepared asdescribed in Example 7 were coupled with various peptides (SEQ IDNOs:2-6) as described, and incubated with murine fibroblasts expressingthe cation-independent mannose-6-phosphate (M6P) receptor overnight. Intwo cases, 2 mM free M6P was added to the culture medium with peptideconjugates (“+M6P”).

TABLE 1 BRIEF DESCRIPTION OF THE SEQUENCES Sequences appearing in theSequence Listing SEQ ID NO Sequence Description SEQ ID NO:1GKKKKKKKKKGC-amide K9 SEQ ID NO:2 CF-GGGYGRKKGGQRRRGGGC- Tat* amide SEQID NO:3 CF-GGGGGKGGGKGGGGGC-amide K2 SEQ ID NO:4CF-GGGKKKKKKKKKGGGC-amide K9 SEQ ID NO:5 CF-GGGkkkkkkkkkGGG-amide k9**SEQ ID NO:6 Biotin-GRRRRRRRRRGC-OH R9 SEQ ID NO:7 THRPPMWSPVWP SEQ IDNO:8 ASSLNIA SEQ ID NO:9 CKGGRAKDC SEQ ID NO:10 GETRAPL *CF -carboxyfluorescein; **k - (D) lysine.

DETAILED DESCRIPTION OF THE INVENTION

Ternary Conjugates

The present invention provides ternary conjugates comprising:

-   -   (1) a therapeutic glycoprotein (G),    -   (2) at least one masking moiety (M) covalently linked to the        glycoprotein through a first linker (L¹), and    -   (3) at least one targeting moiety (T) covalently linked to the        masking moiety through a second linker (L²),        wherein the therapeutic glycoprotein is released from the        conjugate under lysosomal conditions thereby yielding a        biologically active glycoprotein at the site of action in the        cell, e.g., in the lysosome.    -   In one embodiment, the conjugate comprises:    -   (1) a therapeutic glycoprotein (G),    -   (2) at least one masking moiety (M) covalently linked to an        oligosaccharide side chain of the glycoprotein through a first        linker (L¹), and    -   (3) at least one targeting moiety (T) covalently linked to the        masking moiety through a second linker (L²),        wherein the therapeutic glycoprotein is released from the        conjugate under lysosomal conditions thereby yielding a        biologically active glycoprotein at the site of action in the        cell, e.g., in the lysosome.    -   In another embodiment, the conjugate comprises:    -   (1) a therapeutic glycoprotein (G),    -   (2) at least one masking moiety (M) covalently linked to an        amino acid residue of the glycoprotein through a first linker        (L¹), and    -   (3) at least one targeting moiety (T) covalently linked to the        masking moiety through a second linker (L²),        wherein the therapeutic glycoprotein is released from the        conjugate under lysosomal conditions thereby yielding a        biologically active glycoprotein at the site of action in the        cell, e.g., in the lysosome.

The term “biologically active” refers to a function or set of functions(or the effect to which the function is attributed) performed by amolecule in a biological system in vivo or in vitro. Biological activitymay be assessed by, for example, enzymatic activity or inhibitoryactivity as described in the Examples.

The release of the therapeutic glycoprotein may occur as a result ofdegradation of L¹, M, or both at the site of action. Optionally, L² mayalso be degradable at the site of action.

In some embodiments, the ternary conjugates are rapidly degradable atthe site of action in a cell. The term “rapidly degradable” means thatup to 50%, 60%, 70%, 80%, 90%, or substantially all of the glycoproteinis released from the conjugate within 48 hours under lysosomalconditions. (The time can be measured from the time of theadministration to a subject or from the time of intracellular uptake).In such embodiments, the half-life of the conjugate (the time at which50% of the administered glycoprotein is released) is less than 48 hours,e.g., about 6, 12, 18, 24, 30, 36, 42, and 46 hours. The conjugate canbe rapidly degradable due to the masking moiety or a linker, or both.

The term “lysosomal conditions” refers to conditions within thelysosome. The lysosome is a cytoplasmic organelle which, when isolatedunder appropriate conditions, displays one or more lysosomal hydrolaseactivities. Lysosomal isolation procedures are described in e.g.,Bonifacino et al. (eds.) Current Protocols in Cell Biology, John Wiley &Sons, Inc., 2002, section 3.6. In general, the lysosomal conditions maybe reproduced in vitro and include a pH of about 4.5-5.5 and a reducingenvironment as illustrated in the Examples.

In other embodiments, the ternary conjugates are slowly degradable atthe site of action in a cell. The term “slowly degradable” means thatless than 50%, 40%, 30%, 20%, 10% or substantially none of theglycoprotein is released from the conjugate after approximately 48 hoursunder lysosomal conditions. In such embodiments, the half-life of theconjugate is more than 48 hours, e.g., 50, 96, 168, 216, 240, 360, or480 hours. The conjugate can be slowly degradable due to the maskingmoiety or a linker, or both.

The ternary conjugates of the invention may comprise as many as 20masking moieties (M), each independently linked to at least one and asmany as 20 targeting moieties (T). Generally, a conjugate of theinvention or a part thereof has the following formula:G(L¹-M(L²-T)_(n))_(m)  (I)where n and m are integers; and 1≦n≦20 and 1≦m≦20, independently of eachother. n and/or m may, for example, be chosen from 2 to 16, 4 to 12, 1to 8, or 2 to 4. For example, a particular conjugate molecule maycomprise two masking moieties M, with one of the two masking moietiescomprising 4 targeting moieties, while the other masking moiety maycomprise 12 targeting moieties. For illustration purposes only andwithout limitation, FIG. 1 provides a schematic structuralrepresentation of a hypothetical conjugate molecule containing m maskingmoieties and a varying number (q_(m)) of targeting moieties associatedwith each masking moiety (linkers are omitted from the figure). Maskingmoieties M may be the same or different; linkers L¹ may be same ordifferent; targeting moieties T may be the same or different.Additionally, there could be one or more masking moieties that do nothave any L²-T or T attached thereto so long as there is at least, onaverage, one masking moiety that does. Similarly, there could be one ormore L¹ that do not have any M. Thus, the ratio of number of targetingmoieties to the number of masking moieties in a conjugate compositionmay be less than 1, e.g., as low as 0.1. Likewise, the ratio of thenumber of masking moieties per the number of G's in a conjugatecomposition may be less than 1, e.g., as low as 0.1. The embodimentswith n≧2 may provide an additional advantage of “multivalent display” ofthe targeting moiety, which may allow enhanced intracellular uptakeunder some conditions.Glycoprotein

The term “therapeutic glycoprotein” refers to a protein that bears oneor more O- and/or N-linked oligosaccharide side chain(s) such that whenthe glycoprotein is delivered intracellularly, it will exert atherapeutic effect such as, e.g., the prevention, delayed onset, oramelioration of symptoms in a patient or otherwise produce a desiredbiological outcome, such as, e.g., an improved organelle, cell, tissue,or organ function due to, for example, reduced substrate accumulation,reduced cell growth, induction of apoptosis, etc. In some embodiments,the therapeutic glycoprotein is a nonviral glycoprotein, e.g., anantibody. One class of therapeutic glycoproteins is enzymes that aredeficient in a patient to be treated. Examples of such enzymes includelysosomal enzymes such as lysosomal hydrolases listed in Table 2. Insome embodiments, the therapeutic glycoprotein is α-Galactosidase A,acid β-glucosidase (glucocerebrosidase), acid α-glucosidase or acidsphingomyelinase In some embodiments, the therapeutic glycoprotein isα-Galactosidase A, or acid β-glucosidase (glucocerebrosidase).

TABLE 2 Lysosomal Storage Disorders and Corresponding GlycoproteinsDefective enzyme/ Lysosomal storage disorder Therapeutic glycoproteinFabry α-Galactosidase A Farber Acid ceramidase Fucosidosis Acidα-L-fucosidase Gaucher types 1, 2, and 3 Acid β-glucosidase(glucocerebrosidase) G_(M1) gangliosidosis* Acid β-galactosidase HunterIduronate-2-sulfatase Hunter-Scheie α-L-Iduronidase KrabbeGalactocerebrosidase α-Mannosidosis Acid α-mannosidase β-MannosidosisAcid β-mannosidase Maroteaux-Lamy Arylsulfatase B Metachromaticleukodystrophy Arylsulfatase A Morquio A N-Acetylgalactosamine-6-sulfatesulfatase Morquio B Acid β-galactosidase Niemann-Pick Acidsphingomyelinase Pompe Acid α-glucosidase Sandhoff* β-Hexosaminidase BSanfilippo A Heparan N-sulfatase Sanfilippo B α-N-AcetylglucosaminidaseSanfilippo C Acetyl-CoA:α-glucosaminide N-acetyltransferase Sanfilippo DN-Acetylglucosamine-6-sulfate sulfatase Schindler-Kanzakiα-N-acetylgalactosaminidase Sialidosis Sialidase Sly β-GlucuronidaseTay-Sachs* β-Hexosaminidase A *Diseases resulting from the storage ofglycosylceramide-based glycosphingolipids.

The therapeutic glycoprotein may contain two or more subunits (such as,e.g., α-galactosidase A which is a homodimer of two 45 kDa subunits)with one or more of these subunits bearing at least one oligosaccharidechain.

Targeting Moiety

The targeting moiety is selected based on the target cell type, tissue,or organ to allow sufficiently specific delivery of the therapeuticglycoprotein to the desired target. Examples of targeting moietiesinclude:

(1) transducing peptides such as, e.g., R9 (SEQ ID NO:6) (Mitchell etal., J. Peptide Res. (2000) 56:318-325; Wender et al., Proc. Natl. Acad.Sci. (2000) 97:13003-13008), K9 (SEQ ID NO:4) (Shen et al., Proc. Natl.Acad. Sci. (1978) 75:1872-76; U.S. Pat. No. 4,701,521), Tat (SEQ IDNO:2) (Mann et al., EMBO J. (1991) 10:1733-39; Fawell et al., Proc.Natl. Acad. Sci. (1994) 91:664-668; Schwarze et al., Science (1999)285:1569-72), SynB1-SynB6 and sequence variants thereof (Roussele etal., Mol. Pharmacol. (2000) 57:679-686; Day et al., J. Immunol. (2003)170:1498-1503, antennapedia (Derossi et al., Trends in Cell Biol. (1998)8:84-87), VP22 (Elliott et al., Cell (1997) 88:223-233);

(2) natural receptor ligands such as, e.g., insulin (U.S. Pat. No.4,749,570) for targeting through the insulin receptor, insulin-likegrowth factor II (IGF-II) (U.S. Patent Appln. Pub. No. 2003/0082176) fortargeting through the cation-independent mannose 6-phosphate receptor(CIMPR), and receptor-associated protein (RAP) (Prince et al. J. Biol.Chem. (2004) 279:35037-35046) for targeting through LDLR-related protein(LRP), and melanotransferrin (Demeule et al. J. Neurochem. (2002)83:924-933 for targeting through a member of the LDL receptor family tobrain;

(3) phage-display selected peptide ligands such as, e.g., Sp8ca (WO01/90139) for targeting to brain, ASSLNIA (SEQ ID NO:8) (U.S. Pat. No.6,399,575) for targeting to muscle, CKGGRAKDC (SEQ ID NO:9) (Kolonin etal., Nature Med. (2004) 10:625-32) for targeting to adipose tissue,GETRAPL (SEQ ID NO:10) (U.S. Pat. No. 6,399,575) for targeting to muscleor brain; and THRPPMWSPVWP (SEQ ID NO:7) (Lee et al., Eur. J. Biochem.(2001) 268:2004-2012) for targeting through the transferrin receptor tothe brain;

(4) fragments of endogenous proteins such as tissue factor pathwayinhibitor (TFPI) (Hembrough et al., Blood (2004) 103:3374-3380) fortargeting through Very Low Density Lipoprotein (VLDL) receptor;

(5) antibodies to receptors such as, e.g., the anti-transferrin receptorantibody OX26 (Frieden et al., J. Pharm. Exp. Ther. (1996) 278:1491-98;Schnyder et al., Biochem. J. (2004) 377:61-7) and other anti-transferrinreceptor antibodies (Friden et al. 1996; Zhang et al., Mol. Therapy.(2003) 4:1-8 for targeting to the brain; the anti-insulin receptorantibody 83-14hIRMab (Zhang et al., Mol. Therapy. (2003) 7:1-8); theanti-Fc44 antibody (WO 02/057445; Muruganandam et al., FASEB J. (2002)16:240-242);

(6) small molecules such as, e.g., bisphosphonates for targeting tobone; and

(7) non-endogenous proteins and fragments thereof, such as, e.g.,diphtheria toxin CRM₁₉₇, for targeting heparin-binding epidermal growthfactor precursor (HB-EGF) present on the surface of cells in the heartand lung and the blood brain barrier (Gaillard P J, et al. Expert Opin.Drug Deliv. 2005 2(2): 299-309; Abraham et al. BBRC (1993) 190:125-133).

Additional targeting moieties can be made, e.g., as described in Cabilly(ed.), Combinatorial Peptide Library Protocols, 1st ed., Humana Press,1998; Jung (ed.), Combinatorial Peptide and Nonpeptide Libraries: AHandbook, 1997, John Wiley & Sons; and Antibodies: A Laboratory Manual,Harlow et al. (eds.) Cold Spring Harbor Laboratory, 1988; and Borrebaeck(ed.) Antibody Engineering, 2nd ed., 1995, Oxford University Press.

Masking Moiety

A masking moiety is used to mask the oligosaccharide side chain of theglycoprotein from being recognized by its cognate receptor. For example,a masking moiety should be of sufficient size or bulk to reduce (orcompletely block) the binding of the glycoprotein to its cognatereceptor by at least 30%, 40%, 50%, 60%, 70% or more. Suitable methodsfor measuring the reduction or blockage of the glycoprotein-receptor areillustrated in the Examples.

Examples of Masking Moieties Include the Following

(1) non-naturally occurring biocompatible polymers such as, e.g.,polyalkylene oxides, polyethylene glycol (PEG), polyvinylpyrrolidone(PVP), and polymethacrylate (PMA), polyethyleneimine (PEI),polyacrylamide, α,β-poly(N-hydroxyethyl)-DL-aspartamide (PHEA, Cavallaroet al., J. Drug Target. (2004) 12:593-605),poly(vinylpyrrolidone-co-dimethyl maleic anhydride (poly(VP-co-DMMAn)Kamada et al., Nature Biotechnology (2003) 21:399-404),N-(2-hydroxypropyl)methacrylimide) (HMPA, Etrych et al., J. ControlledRelease (2001) 73:89-102.)

(2) polyanionic polysaccharides such as, e.g., polysialic acids (PSA)(e.g., colomininic acid), hyaluronic acid (HA), and dextran sulfate; andhydroxy alkyl starches (e.g., hydroxy methyl starch, hydroxy ethylstarch, hydroxy propyl starch, etc.).

(3) proteinaceous polymers such as, e.g., albumin (e.g., human serumalbumin (HSA), immunoglobulin (IgG).

In some embodiments, the masking moiety is rapidly degradable underlysosomal conditions. The term “rapidly degradable” when used inreference to a masking moiety means that the masking moiety is degradedso that up to 50%, 60%, 70%, 80%, 90%, or substantially all of theglycoprotein is released from the conjugate within 48 hours therebyyielding a biologically active glycoprotein at the site of action in thecell. In such embodiments, the half-life of the release is less than 48hours, e.g., about 6,12, 18, 24, 30, 36, 42, and 46 hours. Rapidlydegradable masking moieties may be synthesized from, e.g., PSA or HA.

In other embodiments, the masking moiety is “slowly degradable” underthe lysosomal conditions, i.e., it is more resistant to degradation andprovides for a more prolonged supply of biologically active glycoproteinat the site of action in the cell. The term “slowly degradable” whenused in reference to a masking moiety means that the masking moiety isdegraded so that less than 50%, 40%, 30%, 20%, 10% or substantially noneof the glycoprotein is released from the conjugate after approximately48 hours under the lysosomal conditions. In such embodiments, thehalf-life of the release is more than 48 hours, e.g., 50, 96, 168, 216,240, 360, or 480 hours. Slowly degradable masking moieties may besynthesized from, e.g., PEG, PVP, or hydroxyethyl starch.

The masking moiety may have a molecular weight of, for example, 0.5-100,1-50, or 10-20 kDa. The masking moiety may comprise one or more (e.g., 2to 40, 2 to 20, 2 to 10, 3, 4, or 5) functional groups for conjugationto the glycoprotein and the targeting moiety. For example, when themasking moiety is PEG, the PEG may comprise multiple arms in either apendant or star configuration, such as a pendant 8- or 16-arm PEG, or astar configuration 4- or 6-arm PEG, or block copolymers containing PEGgroups such as described in, e.g., Funhoff et al., J. Control. Release(2005) 102:711-724; Lecolley et al. Chem. Comm. (2004) 18:2026-2027.

Linkers

The linkers L¹ and L² serve to covalently bind the masking moiety to theglycoprotein and the targeting moiety, respectively. In someembodiments, L¹ and/or L² each comprises a labile group that allows forthe therapeutic glycoprotein to be released from the conjugate underlysosomal conditions thereby yielding a biologically active glycoproteinat the site of action in the cell.

In some embodiments, L¹ and/or L² are/is rapidly degradable underlysosomal conditions. The term “rapidly degradable”, when used inreference to a linker, means that the linker is degraded so that up to50%, 60%, 70%, 80%, 90%, or substantially all of the glycoprotein isreleased from the conjugate within 48 hours thereby yielding abiologically active glycoprotein at the site of action in the cell. Insuch embodiments, the half-life of the release is less than 48 hours,e.g., about 6, 12, 18, 24, 30, 36, 42, and 46 hours. Rapidly degradablelinkers include, for example, hydrazones and disulfides.

In other embodiments, L¹ and/or L² are/is “slowly degradable” under thelysosomal conditions, i.e., they are more resistant to degradation andprovide for a more prolonged supply of biologically active glycoproteinat the site of action in the cell. The term “slowly degradable”, whenused in reference to a linker, means that the linker degraded so thatless than 50%, 40%, 30%, 20%, 10% or substantially none of theglycoprotein is released from the conjugate after approximately 48 hoursunder the lysosomal conditions. In such embodiments, the half-life ofthe release is more than 48 hours, e.g., 50, 96, 168, 216, 240, 360, or480 hours.

The linkers L¹ and L² are each independently chosen preferably fromalkyl (e.g., 1 to 6 carbons), carbonyl, hydrazone, disulfide,heteroaryl, and amido, but additionally may be chosen from alkyl,carbonyl, thiocarbonyl, ether, thioether, ester, disulfide, amino,amido, imino, thioamido, sulfonamido, sulfide, hydrazone, aryl,heteroaryl, cycloalkyl, and heterocyclyl. Any of these groups can beunsubstituted or substituted with one or more functional groups such asaldehyde, alkoxy, amido, amino, aryl, carboxy, cyano, cycloalkyl, ester,ether, halogen, heterocyclyl, hydroxy, ketone, nitro, sulfonate,sulfonyl, or thiol. In some embodiments, alkyl is substituted with acarboxylic acid or an ester thereof. In other embodiments, the ether isa polyether such as a polyalkylene oxide, e.g., polyethylene oxide.

In some embodiments, L¹ and/or L² comprise(s) the hydrazone group offormula (IIa):

In other embodiments, L¹ and/or L² comprise(s) the disulfide group offormula (IIb):

In some embodiments, L¹ and/or L² comprise a hydrazone-containing groupselected from formulas (III)-(VIII):

wherein Ar is aryl, heteroaryl, or pyridyl such as, for example:

In some embodiments, L¹ may contain 0, 1, or 2 hydrazone groups offormula (II) and 0 or 1 disulfide groups, while in the same conjugate L²may contain 0 or 1 hydrazones and 0 or 1 disulfide groups. Examples ofvarious specific embodiments are provided in Table 3. In someembodiments, for example, L¹ and L² each independently include 1, 2, ormore hydrazone groups and additionally a disulfide.

TABLE 3 Examples of The Number and Type of Labile Groups in Linkers L¹and L² L¹ L² Hydrazone —S—S— Hydrazone —S—S— 0 0 0  0* 1 1 1 1 1 1 1 0 11 0 0 1 0 0 0 1 0 1 0 2 1 1 0 2 1 0 1 2 1 1 1 1 0 1 1 1 1 0 1 *In thecase of (0, 0, 0, 0), M is a masking moiety degradable under thelysosomal conditions; in all other cases, this is optional.

For example, L¹ and L² each independently may comprise a group selectedfrom formulas (XIII)-(XX):

wherein p is an integer: 2≦p≦12.

In some embodiments, the conjugates have a formula as shown in(XXI)-(XXV):

In other embodiments, the conjugates have a formula as shown in(XXVI)-(XXXI):

wherein p and q are integers; and 2≦p≦12 and 2≦q≦12 independently ofeach other.

Methods of Making Conjugates

Methods of making conjugates of the invention, including those withformulas (I) and (XVII)-(XVI), comprise: (a) providing a masking moietycomprising a first functional group and a second functional group, (b)reacting the first functional group of the masking moiety with theoligosaccharide side chain of a therapeutic glycoprotein, and (b)reacting the second functional group with a targeting moiety.

The masking moiety may contain a single type of functional group, or itmay be heterofunctional, i.e., it contains at least two different typesof functional groups. For example, the masking moiety may be PEG thatbears any one, any two, or more functional group(s) selected from:hydrazide, hydrazine, amine, hydroxyl, carboxylic acid, ester, thiol,maleimide, acrylate, and vinyl sulfone.

In some embodiments, the methods of making conjugates of the inventioncomprise: (a) reacting an oligosaccharide side chain of glycoprotein Gwith masking moiety M to form glycoprotein-masking moiety conjugate, and(b) reacting targeting moiety T with the glycoprotein-masking moietyconjugate to form ternary conjugate G(L¹-M(L²-T)_(n))_(m).Alternatively, targeting moiety T may be reacted first with maskingmoiety M to form a “ligand-decorated” masking moiety, which is thenreacted with an oligosaccharide side chain of glycoprotein G to formternary conjugate G(L¹-M(L²-T)_(n))_(m).

In some embodiments, an excess molar amount of the masking moiety(ligand-decorated or not) is reacted with the activated glycoprotein(e.g., more than 1, 2, 5, or 10 molar equivalents excess).

Activation of Glycoproteins

In some embodiments, a glycoprotein is activated prior to conjugation byintroducing a reactive group at the linkage site on the oligosaccharideside chain of the glycoprotein. For example, the activated glycoproteinmay bear an electrophilic functional group, e.g., an aldehyde group, atthe linkage site, while the masking moiety (ligand-decorated or not)bears a nucleophilic functional group (e.g., hydrazide) reactive to theelectrophilic group. The activated glycoprotein may further be modifiedto bear a nucleophilic functional group (e.g., a thiol group) byincorporating an adaptor molecule covalently linked to theoligosaccharide, while the masking moiety (ligand-decorated or not) canbe made to bear an electrophilic functional group (e.g., thiol reactivegroup). The thiol-reactive group can be an aryl or heteroaryl disulfide(for example, a pyridyl disulfide). For example, adaptor molecule, suchas nipsylethylamine (NEA; see, e.g., U.S. Pat. No. 6,749,685) orglyoxyl-nipsylethylamide (GNEA) may be reacted with the masking moietyto form a pyridyl disulfide. In other embodiments, the thiolreactive-group can be an aryl or heteroaryl disulfide, vinyl sulfone,vinyl acetate, or maleimide.

Glycoprotein activation may be accomplished either by oxidizing sialicacid and/or other residues (e.g., using periodate), or by first exposinggalactose residues through the removal (“trimming”) of the terminalsialic acid groups (e.g., using an enzyme such as neuraminidase) whichis then followed by oxidation of the exposed galactose to produce aglycoprotein functionalized with an aldehyde (e.g., using an enzyme suchas galactose oxidase (GAO)).

Incorporation of a Hydrazone Group

In some embodiments, an aldehyde is reacted with masking moiety M (see,e.g., FIG. 2 and Example 1) or an adaptor molecule (see, e.g., FIG. 4and Example 7), wherein the masking moiety and the adaptor molecule beara reactive hydrazine group, e.g. a hydrazide. An activated glycoproteinbearing an aldehyde group is exchanged into a buffer with a pH of about5.5(e.g., 5 - 6) and reacted with the hydrazide group. For example, asillustrated in FIG. 2, hydrazide PEG (HzPEG; available commerciallyfrom, e.g., SunBio) is used to form a hydrazone-containing conjugate. Inother embodiments, the adaptor molecule contains a thiol. For example,as shown in FIG. 4, the adaptor molecule 3-(2-pyridyldithio)propionylhydrazide (PDPH) contains a protected thiol. A similar adaptor moleculebearing a hydrazide group, such as S-acetylthioacetimide glutamic acidhydrazide (SATAGH, formed by reacting N-succinimidyl-S-acetylthioacetate(SATA) with glutamic acid γ-hydrazide (SATA; Duncan et al., Anal.Biochem. (1983) 132:68-73) can be used.

The products are then purified away from unreacted PEG or adaptor, e.g.,by anion exchange or size-exclusion chromatography.

Incorporation of a Disulfide Group

One method of attaching a therapeutic glycoprotein and a masking moiety(whether ligand-decorated or not) comprises: (a) incorporating aprotected thiol into an oligosaccharide side chain of an activatedglycoprotein using an adaptor molecule bearing a protected thiol; (b)deprotecting the thiol and reacting it with a thiol-reactive group onthe masking moiety. For example, as illustrated in FIG. 4, aglycoprotein is first reacted with an adaptor molecule bearing aprotected thiol, such as PDPH. Alternatively, an adaptor moleculebearing a protected thiol group may be prepared by reacting SATA withglutamic acid hydrazide to form SATAGH, which may then be reacted with aglycoprotein. The adaptor's thiol group may then be deprotected (e.g.,by reduction of the disulfide with tris-carboxyethylphosphine (TCEP), orby treatment of the thioacetate with hydroxylamine) to expose the thiol,which may then be reacted with a PEG bearing thiol-reactive groups.

The thiol reactive group on the masking moiety may be, for example, adisulfide. In some embodiments, a disulfide may be incorporated into PEGusing an adaptor molecule. For example, HzPEG can be reacted with GNEAto form GNEA-PEG (FIG. 3). Alternatively, a thiol-reactive PEG can bemade by reacting NEA with PEG-bearing carboxylic acid groups (FIG. 4).The resulting conjugate may be purified (e.g., by ion-exchangechromatography) and further reacted with a targeting moiety containing athiol group (e.g., cysteine in a peptide) to yield a final ternaryglycoprotein/masking moiety/targeting moiety conjugate.

Incorporation of Hydrazone and Disulfide Groups

The methods described in the previous two paragraphs can provide linkersbearing both a hydrazone and a disulfide, for example, by using anadaptor molecule comprising both a reactive hydrazide and a protectedthiol. For example, as illustrated in FIG. 4, a glycoprotein is reactedwith the adaptor molecule PDPH, which bears both a protected thiol groupand a hydrazide group. Alternatively, instead of PDPH, a similar adaptormolecule such as SATAGH can be used. As indicated by Duncan et al.,Anal. Biochem. (1983)132, 68-73, the protecting acetyl group in SATA canbe released by hydroxylamine under selective conditions to expose thethiol, which is then available to react with NEA under conditions inwhich less than 20% hydrolysis of the NEA occurs from directhydroxylamine attack.

In other embodiments, such as those illustrated in FIG. 4, an adaptormolecule containing a stabilized hydrazine group such as, e.g., pyridylhydrazide, (and optionally a thiol reactive group) is used to conjugatethe glycoprotein to the masking moiety. Examples of such adaptormolecules include the HydraLink™ reagents (SoluLink Biosciences) asdescribed, e.g., in U.S. Pat. Nos. 5,206,370; 5,420,285; 5,753,520; and5,769,778 and European Patent No. 384,769). For example, PEG-amine canbe reacted with an N-hydroxysuccinimide ester of a pyridyl hydrazine(succinimidyl 4-hydrazinonicotinate acetone hydrazone, “SANH”) orterephthalic acid hydrazide (succinimidyl 4-hydrazidoterephthalatehydrochloride, “SHTH”) to generate a PEG-pyridyl hydrazine or aPEG-terephthalic acid hydrazide. The PEG-pyridyl hydrazine or-terephthalic acid hydrazide can be reacted with a galactose oxidasetreated glycoprotein to yield a hydrazone. Alternatively, a thiolreactive group may be incorporated into the stabilized hydrazine byfirst reacting SANH or SHTH with NEA. The resulting product may bereacted with a masking moiety containing a reactive carbonyl, such as apolyanionic polysaccharide (e.g., PSA) to form a hydrazone.

The glycoprotein/masking moiety conjugate is then further reacted with atargeting moiety. In some embodiments, the targeting group can beconjugated to the masking moiety via a hydrazone linkage as illustratedin FIG. 3. Alternatively, in the case of the targeting moiety being apeptide comprising an N-terminal serine or threonine, the peptide may beoxidized, e.g. with sodium periodate, to yield a glyoxyl peptidederivative. A glycoprotein/masking moiety conjugate comprising ahydrazine may be made, for example, by reacting PEG-amine with SANH orSHTH, followed by reaction with a galactose oxidase treatedglycoprotein. The glyoxyl peptide derivative may then be reacted withthe pyridyl hydrazine or benzoic acid hydrazide groups on the aglycoprotein/masking moiety conjugate to yield a ternary conjugate inwhich the targeting moiety is linked via a hydrazone group. In otherembodiments, a targeting moiety that bears a thiol (e.g., cysteine in apeptide) can be reacted with a thiol-reactive group, for example adisulfide group in an adaptor molecule as illustrated in FIG. 4.

Conjugation with an Amino Acid Residue of a Glycoprotein

In many cases, conjugation of a masking moiety to an oligosaccharidewill produce a high degree of masking of the oligosaccharide andinhibition of receptor binding and clearance. However, as will beappreciated by those of ordinary skill in the art, the distribution ofglycosylation sites in the three-dimensional structure of glycoproteinsmay not always provide an optimal placement for masking criticaloligosaccharide determinants from receptor binding. For example, highmannose oligosaccharides may be uniquely positioned and at a significantdistance from the sites of the complex oligosaccharides, which areamenable to the conjugation chemistry described above.

Accordingly, in another embodiment, methods of making conjugates of theinvention comprise: (a) providing a masking moiety comprising a firstfunctional group and a second functional group, (b) reacting the firstfunctional group of the masking moiety with an amino acid residue of atherapeutic glycoprotein, and (c) reacting the second functional groupwith a targeting moiety.

The methods described above for forming a conjugate of the invention viaan oligosaccharide side chain of the glycoprotein may be also be used toform a conjugate via an amino acid residue of the glycoprotein.

Additionally, in some embodiments, the methods of making conjugates ofthe invention comprise: (a) reacting an amino acid residue ofglycoprotein G with masking moiety M to form a glycoprotein-maskingmoiety conjugate, and (b) reacting targeting moiety T with theglycoprotein-masking moiety conjugate to form ternary conjugateG(L¹-M(L²-T)_(n))_(m). Alternatively, targeting moiety T may be reactedfirst with masking moiety M to form a “ligand-decorated” masking moiety,which is then reacted with an amino acid residue of glycoprotein G toform ternary conjugate G(L¹-M(L²-T)_(n))_(m).

In some embodiments, an amino acid residue is activated prior toconjugation by introducing a reactive group on to the amino acid. Forexample, an amino acid residue of the glycoprotein may be activated tobear a nucleophilic functional group while the masking moiety may bearan electrophilic functional group. In some embodiments, the glycoproteinmay be modified to bear a nucleophilic functional group (e.g., a thiol)by incorporating an adaptor molecule covalently linked to the amino acidresidue, while the masking moiety can be made to bear and electrophilicfunctional group (e.g., a thiol-reactive group). In other embodiments,the amino acid residue may be activated to bear an electrophilicfunctional group, while the masking moiety may bear a nucleophilicgroup.

In some embodiments, an excess molar amount of the masking moiety(ligand-decorated or not) is reacted with the amino acid residue oractivated amino acid residue of a glycoprotein (e.g., more than 1, 2, 5,or 10 molar equivalents excess).

In one embodiment, the amino acid residue to which the masking moiety islinked is a lysine. The lysine may be modified with an adaptor moleculeto introduce a reactive group, such as a thiol. For example, the lysineresidue may be reacted with a thiolation reagent, such as iminothiolane(Traut's reagent), or N-succinimidyl-S-acetylthioacetate (SATA, Duncan,R. J. S. et al. (1983) Anal. Biochem. 132, 68-73). In one embodiment,the thiolation reagent contains spacers, such as SATA-type reagentscontaining a PEG linker such as S-acetyl-dPEG™₄ NHS ester (dPEG™₄ SATA)and S-acetyl-dPEG™₈ NHS ester (dPEG™₈ SATA) (Quanta Biodesign). Thethiol (after deprotection, if necessary) may be reacted with athiol-reactive masking moiety to form a glycoprotein-masking moietyconjugate. For example, the thiol-modified amino acid residue may bereacted with an NEA-PEG to form a disulfide linked glycoprotein-maskingmoiety conjugate (FIG. 4B). The glycoprotein in such disulfide linkedconjugates will be susceptible to release from the masking moiety in thestrongly reducing environment of a lysosome.

In one embodiment, the thiolation reagent is SATA. In anotherembodiment, the thiolation reagent is a SATA-type reagent containing aPEG linker wherein the PEG linker is between 2 and 12 ethylene glycolunits in length, or between 4 and 8 ethylene glycol units in length,such as dPEG™₄ SATA or dPEG™₈ SATA, respectively.

After reaction of the thiol-modified amino acid residue with the thiolreactive masking moiety, the resulting conjugate may be purified, forexample, by ion exchange chromatography. The conjugate may then bereacted with a thiol-containing targeting moiety to form the ternaryconjugate. The targeting moiety may be a peptide or protein containing acysteine residue. Where the targeting moiety does not contain a cysteineresidue, one may be introduced into the protein or peptide sequence or athiol may be introduced by chemical conjugation as described for theglycoprotein. The ternary conjugate may be purified by size-exclusionchromatography or by other means.

Use of a Heterobifunctional Masking Moiety

Yet another method of making ternary conjugates of the inventioninvolves the use of a heterobifunctional masking moiety, for example, amasking moiety which comprises a nucleophile such as, e.g., analdehyde-reactive group, as a first functional group and an electrophilesuch as, e.g., a thiol-reactive group, as second functional group. Sucha method comprises: (a) reacting the heterobifunctional masking moietywith an aldehyde group on the oxidized oligosaccharide side chain of aglycoprotein and (b) reacting a thiol group of a targeting moiety withthe thiol-reactive on the masking moiety. (See, e.g., FIG. 3).

In some embodiments, a heterobifunctional masking moiety is preparedfrom a masking moiety bearing two or more aldehyde-reactive groups,e.g., hydrazide groups (e.g., HzPEG in FIG. 3). As illustrated in FIG.3, a hydrazide-functionalized PEG is reacted with a molecule containinga hydrazide-reactive glyoxyl aldehyde and a thiol-reactive functionalgroup, such as GNEA, to form a heterobifunctional PEG containing ahydrazide group and a thiol reactive group. This heterobifunctional PEGis reacted in the presence of galactose oxidase with aneuraminidase-treated glycoprotein to produce a conjugate in which thePEG is coupled through a hydrazone linkage to an exposed proteinoligosaccharide. This product is purified, and then coupled to a peptidecontaining a free thiol.

Uses of Conjugates

Conjugates of invention can be used as therapeutics in pharmaceuticalcompositions for treatment of mammals (e.g., human and non-humananimals). If necessary, the therapeutic effect of a conjugate may betested using suitable assays such as described in the Examples and/or invivo animal models (e.g., described in Jeyakumar et al., Neuropath.Appl. Neurobiol. (2002) 28:343-357; Mizukami et al., J. Clin. Invest.(2002) 109:1215-1221; Raben et al., J. Biol. Chem. (1998)273(30):19086-92; Marshall et al. Mol. Ther. (2002) 6(2):179-89; Ohshimaet al. Proc. Nat. Acad. Sci. (1997) 94(6):2540-4; Horinouchi et al. Nat.Genet. (1995) 10(3):288-93; McEachern et al. J. Gene Med. (2006 Mar. 10)(Epub ahead of print)). The data obtained from cell culture assays oranimal studies can be used in formulating dosage ranges of for use inhumans. Therapeutically effective dosages achieved in one animal modelcan be converted for use in another animal, including humans, usingconversion factors (e.g., Equivalent Surface Area Dosage factor) knownin the art (see, e.g., Freireich et al. (1966) Cancer Chemother.Reports, 50(4):219-244).

Pharmaceutical compositions will comprise a conjugate of the inventionand one or more suitable pharmaceutical excipients. Variouspharmaceutical excipient formulations are well known (see, e.g.,Physicians' Desk Reference (PDR) 2003, 57th ed., Medical EconomicsCompany, 2002; and Gennado et al. (eds.), Remington: The Science andPractice of Pharmacy, 20th ed, Lippincott, Williams & Wilkins, 2000).

The conjugates of the invention may be used to treat or prevent variousdiseases and disorders including lysosomal storage disorders listed inTable 2. One approach to treating these diseases is enzyme replacementtherapy utilizing the conjugates. In some embodiments, the conjugatesmay be used to treat Fabry, Gaucher, Pompe or Niemann-Pick B disease.

Fabry disease is a rare, inherited lysosomal storage disorder withmultisystemic effects. Patients with Fabry disease have a defect in thegene for the lysosomal enzyme α-galactosidase A (α-gal), also known asceramide trihexosidase. This defect results in an inability ordiminished ability to catabolize lipids with terminal α-galactosylresidues. In the absence of sufficient α-gal, these lipids, particularlyglobotriaosylceramide (GL-3; also known as Gb3, ceramide trihexoside,and CTH), accumulate progressively in the lysosomes of many cell typesthroughout the body. GL-3 accumulation in renal endothelial cells mayplay a role in renal failure.

Gaucher disease is an inherited lysosomal storage disorder. In Gaucherdisease, a deficiency of the enzyme acid β-glucosidase(glucocerebrosidase) leads to the accumulation of the lipidglucocerebroside within the lysosomes of the monocyte-macrophage system.Lipid-engorged cells with eccentric nuclei, known as Gaucher cells,accumulate and displace healthy normal cells in bone marrow and visceralorgans, causing a host of signs, including skeletal deterioration,anemia, hepatosplenomegaly, and organ dysfunction. In rare cases Gauchercells affect the brain and nervous system.

Pompe disease is a debilitating, progressive and often fatal lysosomalstorage disorder. People born with Pompe disease have an inheriteddeficiency of acid α-glucosidase. Acid α-glucosidase assists in thebreakdown of glycogen, a complex sugar molecule stored within thelysosome. In Pompe disease, acid α-glucosidase activity may bedramatically reduced, dysfunctional, or non-existent, resulting in anexcessive accumulation of glycogen in the lysosome. Eventually, thelysosome may become so clogged with glycogen that normal cellularfunction is disrupted and muscle function is impaired. Although there isglycogen storage in the cells of multiple tissues, heart and skeletalmuscles are usually the most seriously affected. Patients typicallyexperience progressive muscle weakness and breathing difficulty, but therate of disease progression can vary widely depending on the age ofonset and the extent of organ involvement.

Niemann-Pick B disease is a lysosomal storage disorder caused bymutations in the gene that encodes a lysosomal enzyme called acidsphingomyelinase (ASM). Due to these mutations, the ASM enzyme is notpresent in sufficient quantities to metabolize fat-like substances. Inpatients with Niemann-Pick B disease, fat-like substances, such assphingomyelin and cholesterol, accumulate in body tissues and organs,resulting in their malfunction. Clinical manifestations of the diseaseare expressed in tissues such as spleen, liver, and lung, and to alesser extent in bone marrow and lymph nodes.

Conjugates of the invention may be administered via any route ofdelivery including parenteral (e.g., subcutaneous, intravenous,intramedullary, intraarticular, intramuscular, or intraperitoneal),transdermal, and oral (e.g., in capsules, suspensions, or tablets). Theconjugates may also be administered by direct administration to thenervous system (e.g. direct injection into the brain,intraventricularly, intrathecally). More than one route can be usedconcurrently, if desired.

Conjugates of the present invention may be administered alone or inconjunction with other agents, such as antihistamines orimmunosuppressants. The term “in conjunction with” indicates that thatthe agent is administered at about the same time as the conjugate. Theagent can be administered contemporaneously or it can be administeredwithin a short time frame (e.g. within 24 hours) of administration ofthe conjugate.

A therapeutically effective amount of the conjugates of the inventionmay be administered at regular intervals depending on the nature andextent of the disease's effect. A therapeutically effective amount is adosage amount that when administered at regular intervals is sufficientto treat the disease such as by ameliorating the symptoms associatedwith the disease, preventing or delaying the onset of the disease and/orlessening the severity or frequency of symptoms of the disease.Effective doses can be extrapolated from dose response curves derivedfrom in vitro and in vivo data. The amount which will be therapeuticallyeffective in the treatment of the disease will depend on the nature andextent of the disease effects and can also be determined by standardclinical techniques. The appropriate therapeutically effective dose willdepend on the route of administration and the seriousness of the diseaseand should be decided by a treating clinician based on each patient'scircumstances. The effective doses can be varied (e.g. increased ordecrease) over time, depending on the needs of the individual.

Most commonly, proteinaceous compounds are administered in an outpatientsetting at regular intervals depending on the nature and extent ofdisease. Administration at a “regular interval” as used herein,indicates that the therapeutically effective dose is administeredperiodocially. The interval an be determined by standard clinicaltechniques. For example, the conjugate is administered daily, weekly,biweekly, monthly, bimonthly, or at longer intervals. The administrationfor a single individual need not be a fixed interval but can varied overtime, depending on the needs of the individual.

EXAMPLES Example 1 Preparation of A Dihydrazide PEG Conjugate ofα-Galactosidase

One milligram of recombinant human α-galactosidase A (α-Gal) in 50 mMsodium phosphate pH 7 was treated overnight with 20 mU/mg Arthrobacter™neuraminidase. A portion (0.5 mg) of the product in 100 μL was incubatedwith 100 μL 10% w/v 10 kDa dihydrazide PEG (Sunbio) and 25 μL 0.2 Msodium succinate pH 5.5 (final pH ˜5.8) overnight at 37° C. with 9 μL of1 mg/mL recombinant Dactylium dendroides galactose oxidase. The productwas dialyzed against 10 mM sodium phosphate pH 7 and applied to a DEAESepharose™ column (Pharmacia) equilibrated with the same buffer andeluted with a gradient from 0 to 0.5 M NaCl in 10 mM phosphate pH 7. Thepeak fraction was concentrated and exchanged into 0.05 M sodiumphosphate pH 7 using 50 kDa MWCO centrifugal ultrafilters (Amicon). Aportion was run on a 4-12% SDS polyacrylamide gel (NuPAGE™, Invitrogen)using a neutral-pH MOPS/SDS running buffer at 200 V for 1 hour, and thegel stained with Coomassie™ blue. This demonstrated approximately equalamounts of mono- and di-PEGylated products as assessed by gel mobility.

Example 2 pH Dependence of Hydrazide PEG Conjugate Stability

Aliquots (4 μL, ˜2 μg) of a DEAE Sepharose™ purified dihydrazideconjugate prepared as described in Example 1 were incubated overnight in45 μL 50 mM buffer (either phosphate, succinate, citrate, or acetate) atbetween pH 7.0 and 5.0 for 14 hours at 37° C., and then concentrated onMicrocon™ 50 centrifugal ultrafilters (Amicon) for 10 minutes at 4° C.The retained volumes were collected and a portion of each run on a SDSpolyacrylamide gel (NuPAGE™, Invitrogen) as described in Example 1. Asshown in FIG. 5, incubation at pH 7 resulted in retention of more than80% of the PEGylated material in PEGylated form (as compared to anunincubated control), while reducing the pH of the solution led to adecrease in the amount of PEGylated material. At pH 5.5, less than 10%of the initial PEGylated material remained. There was a correspondingincrease in the free α-galactosidase.

Example 3 Effect of PEGylation on Binding of αGal to CIMPR In Vitro

Two preparations of α-galactosidase conjugated to either a 4-armdendrimer (“star”) hydrazide PEG (Hz4PEG) or a 8-arm hydrazide PEG(Hz8PEG), each of 10 kDa molecular weight, in which propionyl hydrazidegroups were incorporated into the PEG main chain at random positions(SunBio) were prepared as described in Example 1, except thatconcentration of PEGs in the conjugation step was either 0.5 or 10% w/v.The purified conjugates were assessed for binding to purifiedcation-independent mannose-6-phosphate receptor (CIMPR) by surfaceplasmon resonance (Biacore). Conjugate or unmodified α-galactosidasewere diluted and pumped tangentially across a Biacore flow cell at 20μL/min. The soluble form of CIMPR (lacking the membrane anchor sequence)was conjugated to an activated dextran-coated surface on the opticalface of the cell using NHS chemistry. Binding (assessed by a change inrefractive index) expressed in RU (resonance units), less the RUgenerated by a control cell lacking receptor exposed to the samesolution after 3 minutes was plotted against the concentration ofconjugate. The data (FIG. 6) show that maximum inhibition of binding toreceptor was obtained by prior conjugation of the enzyme with 10% w/v4-arm (Hz4)PEG. Approximately 10-fold higher concentration of PEGconjugate was required to produce the same change in RU as unmodifiedα-galactosidase.

Example 4 In Vivo Uptake of HzPEG/α-Galactosidase Conjugate in FabryMice

A conjugate of α-galactosidase A with a 6-arm star hydrazide PEG (20kDa, SunBio) prepared in a similar fashion as described in Example 1 wasinjected at 1 mg/kg body weight into the tail vein of 4-month-oldα-galactosidase knockout mice, and blood samples (˜100 μL) withdrawn atvarious intervals. Separate sets of mice were either injected withunmodified α-galactosidase or enzyme co-injected with 100 mg/kg yeastmannan (Sigma) to transiently block uptake by mannose receptor. Serumwas prepared and the amount of enzyme remaining in circulationdetermined by dilution and assay using4-methylumbelliferyl-α-D-galactoside (4MU-D-αGal, Sigma) as a substratein the presence of 0.12 M N-acetylgalactosamine to suppressα-galactosidase B activity (Mayes et al., Clin. Chim. Acta (1981)112:247-251) as shown in FIG. 7. Mannan co-injection resulted in atwo-fold increase in the area under the concentration-time curve whereasPEG conjugation resulted in a six-fold increase. The half-life of thelonger-lived PEG conjugate in circulation was approximately 4 hours.

Mice in the same experiment were sacrificed after 8 hours andbiodistribution of the conjugates was determined by enzymatic assay on10% w/v homogenates of the tissues prepared in 0.15% Triton™ X-100, 14.5mM citric acid, 30 mM sodium phosphate pH 4.4 using 4MU-D-αGal assubstrate. The activities in the homogenates were normalized to proteincontent determined by BCA assay (Pierce). The data shown in FIG. 8 showa significant reduction in the uptake in liver by the PEG conjugate.Even after 8 hours, a significant amount of the recovered activity wasstill present in serum.

A portion of the liver was fixed in 4% neutral-buffered formalinovernight at 4° C., embedded in paraffin, and sectioned. Paraffin wasremoved from the mounted sections, which were stained using a monoclonalantibody against the human α-galactosidase at 2.5 μg/mL, and visualizedby a goat anti-mouse horseradish peroxidase secondary antibody usingdiaminobenzidine as substrate. The sections were counterstained withMayers Hematoxylin. Whereas the unmodified control enzyme showed strongstaining of Kupffer cells with some staining of hepatocytes, staining ofKupffer cells was substantially reduced with the PEGylated enzyme, whilehepatocytes stained to a similar extent.

Example 5 Preparation of an αgal Conjugate with a Heterobifunctional PEG

A precursor (BTNEA) was obtained by reaction of 27 mg nipsylethylamine(NEA), 76 mg EDC, 40 mg N-hydroxysuccinimide and 37.4 mg t-BOC-threoninein 93% DMSO with 35 mM imidazole pH6 overnight at 50° C. The product waspurified by reverse-phase chromatography on a C18 column (HigginsTarga), eluting with 0.1% TFA (10 min) followed by a gradient of 0-50%acetonitrile (1%/min) in 0.1% trifluoroacetic acid (TFA). The producteluting at 46 minutes was collected and taken to dryness. The productshowed absorbance peaks similar to NEA (270,353 nm cf. 268,345 nm forNEA), which shifted upon reduction with TCEP (258,308 nm cf. 256,312 nmfor NEA). The product, dissolved in 100 μL DMSO, was deblocked byaddition of 24 μL TFA, and incubated at 50° C. for 15 hours, andpurified by C18 reverse phase chromatography under the same conditions(retention time 26 min). The peak was collected and taken to dryness andthen dissolved in DMSO. The deblocked material was oxidized by reactionwith 25 mM NalO₄ in 50% DMSO-buffered with 0.05 M HEPES pH 7.4 for 15minutes at room temperature, followed by C18 chromatography (productGNEA eluting at 31.8 min).

An 8-arm 20 kDa MW hydrazide pendant PEG (SunBio, 1.5 μmol) in whichpropionic acid hydrazide groups were randomly inserted, was reacted with1.6 μmol GNEA in 50% DMSO, 0.025 M succinate pH 5.6 (total 0.42 mL)overnight at 37° C. The reaction was diluted to 0.8 mL with cold 10 mMsodium phosphate pH 7, and dialyzed against the same buffer and thenagainst water. The product was recovered, lyophilized, and the NEAcontent determined by reduction and absorbance at 346 nm (1.05 mol:molPEG).

Neuraminidase-treated αGal (0.23 mg) was reacted with 10% GNEA-PEG in 50mM succinate pH 5.5 in the presence of 7.5 μg recombinant galactoseoxidase overnight at 37° C. The product was diluted with 10 mL of 10 mMsodium phosphate pH 7 and purified over a column of DEAE Sepharose™ asin Example 1.

A portion of purified GNEA PEG conjugate (25 μg) was reacted with 4.25nmol of fluoresceinated peptides of SEQ ID NO:2 or SEQ ID NO:3 overnightat 4° C. and purified away from unreacted free peptide by elutriation oncentrifugal ultrafilters (Centricon™ 50, Amicon™ Corp). The finalproducts contained 2.1 and 1.6 peptides per conjugate (SEQ ID NO:2 andSEQ ID NO:3, respectively) as determined from the absorbances at 495 nmand 280 nm. A cell uptake experiment such as described in Example 8showed no significant increase in lysate activity after overnightincubation with the conjugate prepared with the peptide of SEQ ID NO:2in the medium. In contrast, incubation with comparable amounts ofneuraminidase-treated αGal produced over 10-fold increase in theactivity of the αGal activity in the lysate.

Example 6 Preparation of Thiol-Reactive Peg

A 16-arm PEG in which pendant propionic acid groups are introduced bycopolymerization (SunBio, 5.25 μmol) was reacted with 126 μmolnipsylethylamine (NEA), 420 μmolN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC), 168 μmolN-hydroxysuccinimide, in 1.6 mL 50% dimethylsulfoxide, 0.1 M imidazolepH 6 overnight at 50° C., yielding a two-phase mixture. Additional 0.1 Mimidazole (1.5 mL) was added and the mixture incubated an additional 3hours at 50° C. The product was purified by extensive dialysis againstwater. The NEA content of the PEG, determined by the absorbance at 350nm, was 9.3 NEA/PEG. A small fraction (<10%) of the PEG prepared in thismanner was bound by DEAE-Separose™ in 10 mM sodium phosphate pH7 buffer.This component was removed from the NEAPEG by passage over DEAESepharose™ prior to use in conjugation reactions.

Example 7 Peptide-PEG Conjugates of αGal using Hydrazide/Thiol Chemistry

An aliquot of α-galactosidase (1.16 mg in 0.25 mL of 50 mM sodiumphosphate pH 7) was treated with 20 mU/mg Arthrobacter™ neuraminidaseovernight at 37° C. The product was then combined with 0.125 mL 0.2 Msuccinate (pH 5.4), 46.5 μg recombinant Dactylium galactose oxidase, and0.05 mL 50 mM 3-(2-pyridyldithio)propionyl hydrazide (PDPH, Pierce) in afinal volume of 0.5 mL, and incubated overnight at 37° C. The productwas then dialyzed overnight against cold 50 mM sodium phosphate pH 7.The product (2.3 mg/mL) was reacted with 10 mMtris-carboxyethylphosphine (TCEP, Pierce) to expose the thiols from theconjugated PDPH for 10 minutes at room temperature followed by 3 roundsof desalting on 50 kDa MWCO centrifugal ultrafilters (Centricon™,Amicon™) with cold degassed buffer. The product was then reacted with 1%(w/v) of a thiol-reactive 16-arm pendant NEA-PEG prepared as describedin Example 6 in the same buffer overnight at 4° C. The reaction wasdialyzed against 10 mM sodium phosphate pH 7, loaded on an DEAESepharose™ column equilibrated with the same buffer, and eluted with0.25 M NaCl in 50 mM phosphate pH 7. The eluate was exchanged into 50 mMsodium phosphate pH 7 on centrifugal ultrafilters. Aliquots (20 μgprotein each) were reacted with FAM- or biotin-labeled peptides (SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, AND SEQ IDNO:6) (3.75 nmol each) overnight in 50 mM sodium phosphate pH 7 and afinal volume of 50 μL and purified by desalting on 50 kDa MWCOcentrifugal ultrafilters using the same buffer. The products showed38-56% of the specific activity of the initial α-galactosidase. Theconjugates prepared with fluoresceinated peptides contained between 1.8and 8.5 peptides per protein. A diagrammatic representation of theprocess is shown in FIG. 4.

Example 8 Peptide-Mediated Cell Uptake of αGal Conjugates In Vitro

Peptide conjugates prepared in Example 7 (each 0.1 nmol/min activityusing p-nitrophenyl-α-D-galactoside as substrate) were incubated with animmortalized wild-type murine fibroblast cell line (TME7, Munier-Lehmannet al., J. Biol. Chem. (1996) 271:15166-15174) in 24-well plates in 0.5mL uptake medium (DMEM/F12 with 5% calf serum, 3% BSA and 25 mM HEPES pH6.7) for 24 hrs in a 37° C. incubator with 5% CO₂. Some of the mediawere supplemented with 2 mM mannose-6-phosphate (M6P) to suppress uptakeby the cation-independent M6P receptor, the major route of uptake of theunmodified enzyme by this cell line. The cells were washed three timeswith PBS pH 6.5 and lysed with 0.5×PBS pH 6.5, 1% Triton™ X-100 withprotease inhibitors (Roche), followed by brief sonication in an ice bathslurry. The activity in the lysates were determined as described inExample 4. Activities present in the lysates normalized to proteincontent as determined by BCA assay (Pierce) are shown in FIG. 3. Theinternalization of the enzyme was verified by staining cells exposed toconjugates under similar conditions using a substrate that produces aninsoluble colored product (X-α-Gal). This showed strong staining in thecytoplasm but not in the nucleus of cells exposed to the R9 (SEQ IDNO:6), Tat (SEQ ID NO:2), and k9 (SEQ ID NO:5) peptide conjugates,indicating that the cell-associated activity was internalized and notbound to the surface of the cells.

Example 9 Use of Thioesters in the Preparation of Hydrazone-LinkedConjugates of Lysosomal Disease Glycoproteins

One micromole of glutamic acid γ hydrazide is converted to the acetoneketal by incubation for 1 hour with 10 μmol acetone in DMSO at roomtemperature. Then 2 μmol N-succinimidyl-5-acetylthioacetate (SATA,Pierce Chemical Co.) is added and the incubation continued overnight atroom temperature. The product, S-acetyl thioacetamide glutamic acidhydrazide (SATAGH), is purified by anion exchange chromatography on QAESephadex™ in 20 mM ammonium formate pH 7, eluting with a gradient to 0.5M ammonium formate pH 7. The A₂₁₅ peak corresponding to the amideproduct is collected and lyophilized. The cargo glycoprotein isseparately desialated by overnight digestion with 20 mU/mg Arthrobacterneuraminidase in 50 mM sodium citrate pH 6, and then treated with 10μg/mg recombinant galactose oxidase overnight in the same buffer. To theoxidized glycoprotein, 5 mM SATAGH is added in 25 mM succinate pH 5.5and the mixture incubated at room temperature for 3 hours. The hydrazoneconjugate is purified by ultrafiltration on a Centricon™ YM-30 filter ordialysis against 25 mM EDTA, 25 mM phosphate pH 7.2. A 10-fold molarexcess of a multiarm NEA-PEG is then added along with 0.2 Mhydroxylamine to deprotect the thiol, and the mixture incubatedovernight at room temperature to generate the disulfide-linked PEGadduct.

Example 10 Use of Pyridyl Hydrazides to Generate Polysialic AcidConjugate of α-Galactosidase

One gram of polysialic acid PSA (colominic acid, 30 kDa avg. MW) isdissolved at 100 mg/ml in 0.1 N NaOH and deacylated by incubation at 37°C. for 4 hours. The product is neutralized by the addition of aceticacid to 0.1 M, and the mixture extracted with chloroform:methanol (3:1).Sodium acetate is added to the aqueous phase (to 0.2 M), and the PSAprecipitated with 3 volumes of ethanol followed by high speedcentrifugation (10,000g for 20 min). The pellet is washed with 85%ethanol and taken to dryness in vacuo. The dried pellet is dissolved to100 mg/ml with water. Separately, an adaptor containing a pyridylhydrazine coupled to nipsylethylamine is generated by reaction of 100μmol acetone-5-succinimidyloxycarbonyl)-pyridine-2-ylhydrazone(SANH)(EMD Biosciences) with 150 μmol nipsylethylamine overnight at roomtemperature in 1 mL DMSO. To 100 mg deacylated polysialic acid (˜3.3μmol) is added 10 μmol SANH-NEA product in 5 mL 0.1 M sodium acetate pH5 and the mixture incubated overnight at room temperature to form thehydrazone. The product is dialyzed against PBS, then water, and thenprecipitated with ethanol as before. The pellet is washed with 85%ethanol, taken to dryness in vacuo, and finally re-dissolved to 100mg/mL in water. This material is conjugated with a reduced PDPHconjugate of aGal prepared as described in Example 7. The product ispurified from unconjugated PSA by diafiltration using a 50 kDa MWCOmembrane, and exchanged into 50 mM sodium phosphate pH 7.

Example 11 Synthesis of a PEG-rha-Gal Conjugate

Fabrazyme® (recombinant human α-galactosidase) was reacted at 5 mg/mLwith a 25:1 molar excess of NHS dPEG™₈ SATA (Quanta Biodesign) for 2hours at 25° C. in 50 mM Na phosphate pH 7. The reaction was quenched bya 100-fold molar excess of Tris HCl pH 7 for 30 minutes and the productpurified by dialysis. This produced 4.8 protected (S-acetyl) thiolgroups per 90 kDa homodimer, as determined by reduction in free lysinecontent of the enzyme by assay with trinitrobenzenesulfonic acid (TNBSA,Pierce). The conjugate was then incubated in 50 mM hydroxylaminehydrochloride, 2.5 mM EDTA, 50 mM Na phosphate pH 7.2 in the presence ofa 30-fold molar excess of a 6-arm (star) 20 kDa NEA-PEG prepared as inexample 6, except substituting a 6-arm star 20 kDa carboxylicacid-terminated PEG (SunBio) as the starting material for the pendant16-arm PEG described in example 6. After two hours at 25° C., thereaction was buffer exchanged into 50 mM Na phosphate pH7, 2.5 mM EDTAand incubated overnight at 25° C. The product was 100% in PEGylated formby SDS PAGE. The product was purified by anion exchange chromatographyon DEAE Sepharose, applying the reaction product in 10 mM Na phosphatepH 7, washing with the same buffer, and eluting with 0.25 M Na phosphatepH 7. The product was then dialyzed into 50 mM Na phosphate pH 7.

A SATA-dPEG₄-conjugate of diphtheria toxin CRM₁₉₇ was separatelygenerated by reaction of a 10-fold molar excess of NHS SATA-dPEG₄-NHS(Quanta Biodesign) with CRM197 (List Biological Laboratories) in 10 mMNa phosphate pH 7.4 for 2 hours at 25° C., and purified byultrafiltration against the same buffer. A 2-fold molar excess of theSATA-dPEG₄-CRM₁₉₇ conjugate was then incubated with theStar-NEA-PEG-α-Gal conjugate in 50 mM hydroxylamine hydrochloride, 2.5mM EDTA, 50 mM Na phosphate pH 7.2 for 2 hours at 25° C. to deprotectthe linker-introduced thiols on the CRM₁₉₇. The mixture was then bufferexchanged into 2.5 mM EDTA, 50 mM Na phosphate pH7 and incubatedovernight at 25° C. in the same buffer to complete the couplingreaction. The product was purified by size-exclusion chromatography onSuperdex 200 resin in 50 mM Na phosphate pH 7. The PEGylated producteluted as a single HMW peak which had no free acid galactosidase orCRM₁₉₇. Upon reduction, SDS PAGE analysis showed the presence of CRM₁₉₇to acid galactosidase subunits in approximately 1:1 ratio.

Example 12 Vero Cell Uptake of CRM PEG-α-Gal Conjugate

The CRM conjugate prepared as in example 11 or the star PEG-α-Galconjugate without CRM is added (5 μg/mL) to separate aliquots 0.5 mLuptake medium (Vero cell growth medium buffered with 10 mM HEPES pH 6.7)and applied to Vero cells. To block uptake mediated by thecation-independent mannose-6-phosphate (M6P) receptor, 2 mM M6P is addedto the medium in some wells. In additional control wells, free CRM₁₉₇ isadded at a 10-fold excess over the conjugate, and other wells receive noconjugate to assess the endogenous level of α-galactosidase activity.The plate is placed in an incubator overnight at 37° C., 5% CO₂. Thecell layers are washed 3 times with 1 mL PBS and lysed with ½×PBS pH6.5, 1% Triton X-100, 1× protease inhibitor cocktail (“Complete”,Roche). The lysates are then assayed for acid α-galactosidase activityusing 4-MU-α-galactoside substrate (Sigma) normalized to total proteincontent obtained by BCA assay (Pierce). The activity in the controlwells receiving no protein (endogenous activity) is subtracted from theactivity in the wells which received protein to determine uptake. Theextent of masking is determined from the amount of uptake which issensitive to M6P in the medium without and with PEGylation and/or CRMconjugation. CRM dependent uptake is determined from the α-galactosidaseuptake observed in the wells containing the CRM conjugate less theamount of uptake observed in wells containing excess CRM.

All publications and patents cited in this disclosure are incorporatedby reference in their entirety. To the extent the material incorporatedby reference contradicts to or is inconsistent with this disclosure, theinstant disclosure supersedes. All examples are provided forillustration only and are nonlimiting. Further, all numbers expressingreaction parameters are approximate, unless expressly indicated or thecontext requires otherwise.

The invention claimed is:
 1. A conjugate comprising: (1) a therapeuticglycoprotein (G), (2) a masking moiety (M) capable of reducing orblocking binding of the glycoprotein to its cognate receptor, whereinthe masking moiety is covalently linked to the glycoprotein through afirst linker (L¹), and (3) a targeting moiety (T) covalently linked tothe masking moiety through a second linker (L²), wherein: the conjugatehas formula G(L¹−M(L²−T)_(n))_(m), wherein2≦m≦16, andn <1 or 1 <n ≦20, and the glycoprotein is released from the conjugateunder lysosomal conditions.
 2. The conjugate of claim 1, wherein M iscovalently linked to an oligosaccharide side chain of the glycoproteinthrough L¹.
 3. The conjugate of claim 1, wherein M is covalently linkedto an amino acid residue of the glycoprotein through L¹.
 4. Theconjugate of claim 3, wherein the amino acid residue is a lysineresidue.
 5. The conjugate of claim 1, wherein the glycoprotein is anon-viral protein chosen from an enzyme and an antibody.
 6. Theconjugate of claim 5, wherein the enzyme is a lysosomal enzyme.
 7. Theconjugate of claim 6, wherein the lysosomal enzyme is chosen fromα-galactosidase A, acid ceramidase, acid α-L-fucosidase,glucocerebrosidase, acid β-galactosidase, iduronate-2-sulfatase,α-L-iduronidase, galactocerebrosidase, acid α-mannosidase, acidβ-mannosidase, arylsulfatase B, arylsulfatase A,N-acetylgalactosamine-6-sulfate sulfatase, acid sphingomyelinase, acidα-glucosidase, β-hexosaminidase B, heparan N-sulfatase,β-N-acetylglucosaminidase, cetyl-CoA:α-glucosaminiden-acetyltransferase,N-acetylglucosamine-6-sulfate sulfatase, α-N-acetylgalactosaminidase,sialidase, β-glucuronidase, and β-hexosaminidase A.
 8. The conjugate ofclaim 7, wherein the lysosomal enzyme is chosen from acid α-glucosidase,acid sphingomyelinase, α-galactosidase A, and glucocerebrosidase.
 9. Theconjugate of claim 1, wherein the masking moiety is degradable underlysosomal conditions.
 10. The conjugate of claim 1, wherein the maskingmoiety comprises 2-10 functional groups for conjugation to theglycoprotein and the targeting moiety.
 11. The conjugate of claim 10,wherein the masking moiety comprises 3, 4, or 5 functional groups forconjugation to the glycoprotein and the targeting moiety.
 12. Theconjugate of claim 1, wherein n <1.
 13. The conjugate of claim 1,wherein 1<n ≦20.
 14. The conjugate of claim 1, wherein the maskingmoiety is a polymer chosen from polyethylene glycol (PEG),polyvinylpyrrolidone (PVP), polymethacrylate (PMA), polysialic acid(PSA), hyaluronic acid (HA), albumin, immunoglobulin (IgG), dextransulfate, polyethyleneimine (PEI), polyacrylamide,α,β-poly(N-hydroxyethyl)-DL-aspartamide (PHEA),poly(vinylpyrrolidone-co-dimethyl maleic anhydride (poly(VP-co-DMMAn),N-(2-hydroxypropyl)methacrylimide)(HMPA), and hydroxy alkyl starch(HAS).
 15. The conjugate of claim 14, wherein the masking moiety is aPEG chosen from a star-PEG and a pendant-PEG.
 16. The conjugate of claim14, wherein the masking moiety is a polysialic acid (PSA).
 17. Theconjugate of claim 16, wherein the masking moiety is a polysialic acid(PSA) comprising 2-10 functional groups for conjugation to theglycoprotein and the targeting moiety.
 18. The conjugate of claim 1,wherein the targeting moiety is a transducing peptide, a non-endogenousprotein, a receptor-binding peptide, an antibody to a receptor, or anatural receptor ligand.
 19. The conjugate of claim 1, wherein at leastone of L¹ and L² is amido.
 20. The conjugate of claim 1, wherein atleast one of L¹ and L² comprises at least one group that is labile underlysosomal conditions.
 21. The conjugate of claim 20, wherein L¹comprises at least one labile group.
 22. The conjugate of claim 20,wherein at least one of L¹ and L² comprises at least one acid-labilegroup.
 23. The conjugate of claim 22, wherein the at least oneacid-labile group is chosen from hydrazone, imino, and ester.
 24. Theconjugate of claim 23, wherein the at least one acid-labile group is ahydrazone group.
 25. The conjugate of claim 20, wherein at least one ofL¹ and L² comprises a disulfide.
 26. The conjugate of claim 25, whereinat least one of L¹ and L² comprises a formula chosen from:

wherein:2≦p≦12, and one or more of the terminal atoms of the formula may bederived from the element G, M, or T to which the atoms are attached. 27.The conjugate of claim 25 wherein the disulfide of L^(l), L², or both isformed with the terminal sulfur atom of a formula selected from:

and a sulfur atom of G, M, or T.
 28. The conjugate of claim 20, whereinat least one of L¹ and L² comprises at least one acid-labile group and adisulfide.
 29. The conjugate of claim 28, wherein at least one of L¹ andL² comprises a formula chosen from:

wherein one or more of the terminal atoms of the formula may be derivedfrom the element G, M, or T to which the atoms are attached.
 30. Theconjugate of claim 20, wherein L¹ comprises the formula

wherein 2≦p≦12, and L² comprises the formula

wherein 2≦q≦12.
 31. The conjugate of claim 1, wherein the conjugatecomprises a formula chosen from:

wherein any one or more of the terminal atoms of the formula betweenelements G and M and between elements M and T may be derived from theelement to which the atoms are attached.
 32. A pharmaceuticalcomposition comprising the conjugate of claim 1 and a pharmaceuticallyacceptable excipient.